1. Field of the Invention
The present invention is in the fields of molecular biology, cancer biology and medical therapeutics. The invention is generally directed to the identification of genes that inhibit the growth of, and induce apoptosis in, cancer cells, and to polypeptides encoded by such genes. In particular, the invention concerns the nucleotide sequence of one such tumor-inhibiting gene, tag7, the amino acid sequence of a polypeptide encoded by tag7, antibodies that specifically bind to the tag7 gene product, and methods of inhibiting cancer cell growth and of cancer therapy using the tag7 gene and gene product.
2. Related Art
Tumor Metastasis
The metastasis of tumor cells is a complex process which includes a complex cascade of events. Included among these events are the proliferation of tumor cells and blocking of the apoptosis mechanism (Harrington, E. A., et al., EMBO J. 13:3286-3295 (1994)), the spread of the tumor cells into surrounding tissues, the penetration of tumor cells into the blood and lymphatic circulations, and the attachment and multiplication of tumor cells at a new site (Schirmacher, A. E., Adv. Cancer Res. 43:1-42 (1985); Liotta, L. A., et al., Lab. Invest. 49:636-649 (1983)). While the molecular mechanisms of the induction of the metastatic phenotype remain poorly studied, it is likely that activation and/or inactivation of a variety of regulatory and structural genes occur as a tumor becomes metastatic. Several genes whose expression correlates with the metastatic potential of tumors have been described (Yasushi, T., et al., J. Biol. Chem. 269(37):22958-22963 (1994); Ebralidze, A. K., et al., Genetika (USSR) 25(5):932-936 (1989); Wolf, C., et al., Proc. Natl. Acad. Sci. USA 90:1843-1847 (1993); Bernhard, E. J., et al., Proc. Natl. Acad. Sci. USA 91:4293-4297 (1994); Sato, H., et al., Nature 370:61-65 (1994)).
It has been suggested that the population of cells within a given tumor may be heterogeneous with respect to their metastatic potential (Fidler, I. J., and Hart, I. R., Science 217:998-1001 (1982)). This suggestion offers the possibility of obtaining related tumors that differ markedly in metastatic potential from one parent tumor. For example, tumors that are transplantable to mice and that have a varying frequency and organ-specificity of metastasis have been obtained (Senin, V. M., Vestn. Akad. Med. Nauk. SSSR 0(5):85-91 (1984)) as a result of selection for the metastasis character.
Tumor-inhibiting Polypeptides
A number of polypeptides naturally produced by mammalian cells have been shown to have anti-tumor activity (i.e., inducing growth arrest, apoptosis and/or differentiation of cancer cells). For example, combinations of certain cytokines, such as interleukins and colony-stimulating factors, have been reported to induce terminal differentiation and concomitant growth arrest of particular types of tumor cells and cell lines (reviewed by Pimental E., in: Handbook of Growth Factors, Vol. 1, Boca Raton, Fla.: CRC Press, pp. 28-34 (1994)). In addition, transforming growth factor xcex2 (TGF-xcex2) and the interferons are known to be potent growth inhibitors for tumor cells under certain conditions in vivo and in vitro (Keski-Oja, J., and Moses, H. L., Med. Biol. 66:13-20 (1987); Ohta, M., et al., Nature 329:539-541 (1987)). Other natural mammalian polypeptides having a variety of tumoricidal activities, such as the induction of apoptosis in certain tumor cells, include the tumor necrosis factors (TFNs) (reviewed by Pimental, E., in: Handbook of Growth Factors, Vol. 3, Boca Raton, Fla.: CRC Press, pp. 241-278 (1994)).
Recently, a family of TNF-related cytokines and their receptors with somewhat common structural features has been identified. This family includes the TNF, LT-xcex1, LT-xcex2, Fas, CD27, CD40, OX-40, and nerve growth factor (NGF) receptor-ligand systems (Smith, C., et al., Cell 76:959-962 (1994); Armitage, R. J., Curr. Opin. Immunol. 6:407-413 (1994)). With the exception of NGF, all of these TNF-related cytokines are thought to be involved in the regulation of the immune System TNF and lymphotoxin-alpha (LT-xcex1 or TNF-xcex2) are related cytokines involved in many regulatory activities (Vassalli, P., Ann. Rev. Immunol. 10: 411-452 (1992); Paul, N., and Ruddle, N., Ann. Rev. Immunol. 6:407-438 (1988)) but their roles in the immune system, while apparently critical, remain enigmatic (de Kossodo, S., et al., J. Exp. Med. 176:1259-1264 (1992)). TNF is synthesized in response to various insults by a variety of cell types; this is generally regarded as one of the primary events in the inflammatory cascade, including a potent antitumor effect on mice (Blankenstein, T., et al., J. Exp. Med 173:1047-1052 (1991)). Activated macrophages are the major source of membrane-bound TNF, although it is also produced by activated lymphocytes and several other cell types. In contrast, LT-xcex1 is produced specifically by lymphocytes and exists in membrane-associated form only via a trimeric complex with LT-xcex2 (Browning, J., et al., Cell 72:847-855 (1993)). LT-xcex2 shows a spectrum of activities similar to that of TNF in in vitro systems, but is less potent (Browning, J., and Ribolini, A., J. Immunol. 143:1859-1867 (1989)). Both TNF and LT-xcex1 induce apoptosis in various systems (Cohen, J. J., et al., Ann. Rev. Immunol. 10:267-293 (1992); Golstein, P., et al., Immunol. Rev. 121:29-65 (1991); Sarin, A., et al., J. Immunol. 155:3716-3718 (1995)). Recently, tumor growth inhibition mediated by LT-xcex1 has been reported (Qin, Z., and Blankenstein, T., Cancer Res. 55:4747-4751 (1995)).
TNF and LT-xcex1 are also released by a number of tumor cells of various origins, such as mouse fibrosarcomas, human epithelial cell lines and T-cell leukemia cells and cell lines (Rubin, B. Y., et al., J. Exp. Med. 164:1350-1355 (1986); Spriggs, D. R., et al., J. Clin. Invest. 81:455-460 (1988); Ishibashi, K., et al., Blood 77:2451-2455 (1991)). The genes encoding TNF, LT-xcex1 and LT-xcex2 lie closely spaced within the class III region of the major histocompatibility complex (MHC) (Spies, T., et al., Proc. Natl. Acad. Sci. USA 83:8699-8702 (1986); Nedospasov, S. A., et al., Nucl. Acids Res. 14:7713-7725 (1986); Gardner, S. M., et al., J. Immunol. 139:476-483 (1987)). These genes are thought to be evolutionarily related and are though to have formed the locus by tandem gene duplications, although the opposite orientation of LT-xcex2 transcription suggests that more complex evolutionary events may have taken place. Recently, another novel TNF family member has been cloned and designated as TNF-related apoptosis-inducing ligand (TRAIL) (Wiley, S. R., et al., Immunity 3:673-682 (1995)).
The present invention generally relates to tumor-inhibiting genes and polypeptides, and methods of treating cancers using these genes and polypeptides. Specifically, the invention provides isolated tag7 nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least about 65% (more preferably at least about 70%, at least about 75%/o, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%) identical to a reference sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO:1;
(b) a nucleotide sequence encoding the tag7 polypeptide having the complete amino acid sequence set forth in SEQ ID NO:2;
(c) the nucleotide sequence encoding the mature tag7 polypeptide having the amino acid sequence at positions about 20-182 in SEQ ID NO:2;
(d) a nucleotide sequence of a polynucleotide which hybridizes under stringent conditions to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO:1;
(e) a nucleotide sequence of a polynucleotide which hybridizes under defined conditions to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO:1; and
(f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d) or (e), or a fragment thereof.
The invention is also directed to an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence identical to that of the isolated nucleic acid molecules described above, which may or may not encode a polypeptide having tag7 activity.
The invention is also directed to an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under defined conditions to a polynucleotide having a nucleotide sequence identical to that of the isolated nucleic acid molecules described above, which may or may not encode a polypeptide having tag7 activity.
In a preferred embodiment, the invention is directed to isolated tag7 nucleic acid molecules comprising a polynucleotide having
(a) the nucleotide sequence set forth in SEQ ID NO;3;
(b) a nucleotide sequence encoding the human tag7 polypeptide having the complete amino acid sequence set forth in SEQ ID NO:4;
(c) a nucleotide sequence of a polynucleotide which hybridizes under stringent conditions to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO:3;
(d) a nucleotide sequence of a polynucleotide which hybridizes under defined conditions to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO:3; and
(e) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c) or (d), or a fragment thereof.
The invention also provides vectors, particularly expression vectors, comprising these isolated nucleic acid molecules, and host cells comprising these isolated nucleic acid molecules or vectors. Preferred host cells of the invention include, but are not limited to, bacterial cells, yeast cells, animal cells (especially mammalian cells or insect cells) and plant cells.
The invention also relates to methods for producing an isolated tag7 polypeptide, comprising culturing the above-described host cells under conditions sufficient to allow the expression of a tag7 polypeptide, and isolating the polypeptide. The invention is also directed to isolated tag7 polypeptides produced according to these methods.
The invention also is directed to isolated tag7 polypeptides having an amino acid sequence at least about 65%(more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%) identical to a reference sequence selected from the group consisting of:
(a) the amino acid sequence encoded by an isolated nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO:1;
(b) the complete amino acid sequence of the tag7 polypeptide as set forth in SEQ ID NO:2;
(c) the amino acid sequence of a tag7 polypeptide encoded by a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:1; and
(d) the amino acid sequence of a tag7 polypeptide encoded by a polynucleotide which hybridizes under defined conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:1, or a fragment thereof
In a preferred embodiment, the invention is directed to a human tag7 polypeptide having
(a) the amino acid sequence encoded by an isolated nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO:3;
(b) the amino acid sequence of the tag7 polypeptide as set forth in SEQ ID NO:4; or a fragment thereof.
The invention also relates to pharmaceutical compositions comprising one or more of the above-described isolated tag7 polypeptides or fragments thereof and a pharmaceutically acceptable carrier or excipient therefor.
The invention also relates to methods of producing an isolated tag7-specific antibody comprising immunizing an animal with the above-described isolated tag7 polypeptides, and isolating a tag7-specific antibody from the animal. The invention is also directed to isolated tag7-specific antibodies produced by these methods. The antibodies of the invention may be polyclonal or monoclonal antibodies, and may be detectably labeled or immobilized on a solid support.
The invention also relates to methods of inhibiting the growth of a mammalian tumor, such as a human tumor. In one preferred embodiment, such methods of the invention may comprise contacting a mammalian cell with a composition comprising one or more isolated tag7 polypeptides, wherein the isolated tag7 polypeptide has an amino acid sequence at least about 65% identical (more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% identical) to a reference sequence selected from the group consisting of:
(a) the amino acid sequence encoded by an isolated nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO:1;
(b) the complete amino acid sequence of the tag7 polypeptide as set forth in SEQ ID NO:2;
(c) the amino acid sequence of the mature tag7 polypeptide having the amino acid sequence as set forth at positions 20 to 182 in SEQ ID NO:2;
(d) the amino acid sequence encoded by a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:1; and
(e) the amino acid sequence encoded by a polynucleotide which hybridizes under defined conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:1,
wherein the contacting of the mammalian cell with the tag7 polypeptide inhibits the growth of the cell.
In a preferred embodiment of this method, the mammalian tumor is a human tumor, and human cells are contacted with a composition comprising human tag7 polypeptide having the amino acid encoded by an isolated nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO:3; or a fragment thereof.
In another preferred embodiment, the invention relates to methods of inhibiting the growth of a mammalian tumor comprising introducing into a mammalian cell a nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least about 65% (more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%) identical to a reference sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO:1;
(b) a nucleotide sequence encoding the tag7 polypeptide having the complete amino acid sequence set forth in SEQ ED NO:2;
(c) a nucleotide sequence encoding the mature tag7 polypeptide having the amino acid sequence at positions 20 to 182 in SEQ ED NO:2;
(d) the nucleotide sequence of a nucleic acid molecule encoding a tag7 polypeptide, comprising a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:1; and
(e) the nucleotide sequence of a nucleic acid molecule encoding a tag7 polypeptide, comprising a polynucleotide which hybridizes under defined conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:1,
wherein the introduction of the isolated nucleic acid molecule into the mammalian cell inhibits the growth of the tumor.
In a preferred embodiment, the invention relates to methods of inhibiting the growth of a human tumor comprising introducing into a human cell a nucleic acid molecule comprising the human tag7 polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 3 or a fragment thereof.
According to the invention, the above-described tumor may be induced to undergo apoptosis through the action of the present methods. Preferred mammalian tumors inhibited from growing according to the methods of the invention include human tumor cells, particularly carcinoma cells (including but not limited to liver carcinoma cells, ovarian carcinoma cells, breast carcinoma cells, cervical carcinoma cells, lung carcinoma cells, prostatic carcinoma cells, gastric carcinoma cells, bladder carcinoma cells, testicular carcinoma cells, colorectal carcinoma cells, pancreatic carcinoma cells, oral cavity carcinoma cells, squamous cell carcinoma cells, head and neck carcinoma cells and teratocarcinoma cells), sarcoma cells (including but not limited to Kaposi""s sarcoma cells, fibrosarcoma cells and osteosarcoma cells), melanoma cells and leukemia cells.
The invention also relates to methods for treating a cancer in an animal (particularly a mammal such as a human) suffering therefrom. In one preferred embodiment, such methods may comprise administering to the animal a composition comprising one or more isolated tag7 polypeptides, wherein the isolated tag7 polypeptide has an amino acid sequence at least about 65% (more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%) identical to a reference sequence selected from the group consisting of:
(a) the amino acid sequence encoded by an isolated nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO:1;
(b) the complete amino acid sequence of the tag7 polypeptide as set forth in SEQ ID NO:2;
(c) the amino acid sequence of the mature tag7 polypeptide having the amino acid sequence as set forth at positions 20 to 182 in SEQ ID NO:2;
(d) an amino acid sequence encoded by an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO.1; and
(e) an amino acid sequence encoded by an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under defined conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:1,
wherein the treatment inhibits the progression or growth, or induces the remission, of the cancer.
In another preferred embodiment, such methods of the invention may comprise introducing into the animal a nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least about 65% (more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%) identical to a reference sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ D) NO:1;
(b) a nucleotide sequence encoding the tag7 polypeptide having the complete amino acid sequence set forth in SEQ ID NO:2;
(c) a nucleotide sequence encoding the mature tag7 polypeptide having the amino acid sequence at positions 20 to 182 in SEQ ID NO:2;
(d) the nucleotide sequence of a polynucleotide which hybridizes under stringent conditions to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO:1; and
(e) the nucleotide sequence of a polynucleotide which hybridizes under defined conditions to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO:1,
wherein the treatment inhibits the progression or growth, or induces the remission, of the cancer.
In a preferred embodiment, such methods may comprise administering to a human a composition comprising an isolated human tag7 polynucleotide having a nucleotide sequence set forth in SEQ ID NO: 3, or one or more fragments thereof.
In another preferred embodiment, the invention relates to methods of treating a cancer in an animal suffering therefrom comprising administering to the animal one or more of the above-described pharmaceutical compositions comprising one or more isolated tag7 polypeptides of the invention.
According to the invention, the isolated tag7 polypeptides used in the above-described methods preferably have amino acid sequences at least 95% identical to the above-described reference sequences. The tag7-containing compositions used in the above-described methods may further comprise a pharmaceutically acceptable carrier or excipient for the isolated tag7 polypeptide.
Also according to the invention, the polynucleotides used in the above-described methods preferably have nucleic acid sequences at least about 65% (more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%) identical to the above-described reference sequences, and may be contained in a vector or a virion, which may be derived from an adenovirus or an adeno-associated virus.
The animal suffering from the cancer treated by the methods of the invention may be a mammal such as a human. The cancer treated by these methods may include, without limitation, a carcinoma (such as a a liver carcinoma, an ovarian carcinoma, a breast carcinoma, a cervical carcinoma, a lung carcinoma, a prostatic carcinoma, a gastric carcinoma, a bladder carcinoma, a testicular carcinoma, a colorectal carcinoma, a pancreatic carcinoma, an oral cavity carcinoma, a squamous carcinoma, a head and neck carcinoma or a teratocarcinoma), a sarcoma (such as a Kaposi""s sarcoma, a fibrosarcoma or an osteosarcoma), a melanoma or a leukemia.
Other preferred embodiments of the present invention will be apparent to one of ordinary skill in light of the following drawings and description of the invention, and of the claims.